The Effect of N-linked Glycosylation on Molecular Weight, Thrombin Cleavage, and Functional Activity of Human Protein S
The Effect of N-linked Glycosylation on Molecular Weight, Thrombin Cleavage, and Functional Activity of Human Protein S
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N 连接糖基化对人蛋白 S 的分子量、凝血酶裂解和功能活性的影响
DOI：
10.1055/s-0038-1656130
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发表时间：
1997
影响因子：
6.7
通讯作者：
G. Long
中科院分区：
文献类型：
--
作者：
D. Lu;Rongze Xie;A. Rydzewski;G. Long
人类蛋白S（HPS）在Asn 458 '468' 489处具有三个潜在的N-连接糖基化位点。为了研究这些位点糖基化的作用，采用PCR突变法消除全长HPS cDNA中每个N-连接糖基化位点（Asn458 → Gln，Ser460 → Gly; Asn468 → Gln，Thr470 → Gly; Asn489 → Gln，Thr491 → Gly）的共有序列。通过稳定转染cDNA/SV40/adeno/pBR322衍生的表达载体，在人肾293细胞中表达每个所得的构建体，收集条件培养基用于重组蛋白纯化。SDS-PAGE凝胶显示，糖基化突变体的迁移相同，比野生型rHPS更快，表明每个三个潜在的N-糖基化位点含有类似量的碳水化合物。质谱分析得到了类似的结果，野生型HPS的分子量约为78，000。为了证明野生型和突变蛋白S之间的迁移率差异是由于它们的碳水化合物含量，使血浆来源的HPS和重组HPS经受N-聚糖酶消化，随后显示在SDS-PAGE凝胶上相同地迁移。所有形式的HPS被α-凝血酶裂解的时间过程相似。功能研究表明，野生型rHPS具有相同的辅因子的具体活动，血浆衍生的HPS，通过标准的凝血试验测试。Asn458和Ser460突变体rHPS仅具有略高的辅因子活性，而其他四种突变体与野生型rHPS相比具有相似的凝血活性。在一个纯化的组件系统中，蛋白S的糖基化突变体表现出轻微增强的能力，刺激APC介导的因子Va失活后的初始滞后期。采用液相平衡结合实验研究了重组HPS糖基化突变体与人C4b结合蛋白（C4bp）的相互作用。两个突变体（Asn458，Ser460）与C4bp的解离常数（Kd）略低，而其他突变体与野生型rHPS具有相同的表观Kd。
Summary Human protein S (HPS) has three potential N-linked glycosylation sites at Asn458’468’489. To study the role of glycosylation at these sites, PCR mutagenesis was used to abolish the consensus sequence of each N-linked glycosylation site (Asn458→Gln, Ser460→Gly; Asn468→Gln, Thr470→Gly; Asn489→Gln, Thr491→Gly) in full-length HPS cDNA. Each resulting construct was expressed in human kidney 293 cells by stable transfection of cDNA/SV40/adeno/pBR322-derived expression vectors, and conditioned medium was collected for recombinant protein purification. SDS-PAGE gels revealed that glycosylation mutants migrate identically and faster than the wild-type rHPS, showing that each of the three potential N-glycosylation sites contain a similar amount of carbohydrate. Mass spectral analysis yielded similar results and a molecular mass of ~78,000 for wild-type HPS. To demonstrate that the difference in mobility between wild-type and mutant protein S is due to their carbohydrate content, plasma-derived HPS and recombinant HPS were subjected to N-glycanase digestion and subsequently shown to migrate identically on SDS-PAGE gels. All forms of HPS have similar time courses for cleavage by α-thrombin. Functional studies indicate that wild-type rHPS possesses the same cofactor specific activity as plasma-derived HPS, as tested by a standard clotting assay. Asn458 and Ser460 mutant rHPS have only a slightly higher cofactor activity, whereas the other four mutants have similar clotting activities, compared to wild-type rHPS. In a purified component system, glycosylation mutants of protein S showed a slightly enhanced ability to stimulate APC-mediated factor Va inactivation after an initial lag phase. The interaction of rHPS glycosylation mutants with human C4b-binding protein (C4bp) was also studied by solution phase equilibrium binding assay. Two mutants (Asn458, Ser460) have marginally lower dissociated constants (Kd) with C4bp, whereas the others have the same apparent Kd as wild-type rHPS.
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DOI：
10.1172/jci114926
发表时间：
1990
期刊：
The Journal of clinical investigation
影响因子：
--
作者：
Weinstein,RE;Walker,FJ
通讯作者：
Walker,FJ
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影响因子：
20.3
作者：
Schmidel,DK;Nelson,RM;BroxsonJr,EH;Comp,PC;Marlar,RA;Long,GL
通讯作者：
Long,GL
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DOI：
--
发表时间：
1993
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Fernández,JA;Heeb,MJ;Griffin,JH
通讯作者：
Griffin,JH
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DOI：
10.1042/bj3050397
发表时间：
1995
期刊：
The Biochemical journal
影响因子：
--
作者：
Greengard,JS;Fernandez,JA;Radtke,KP;Griffin,JH
通讯作者：
Griffin,JH
活化的蛋白 C 与因子 V 和 Va 的结合。
DOI：
--
发表时间：
1986
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Krishnaswamy,S;Williams,EB;Mann,KG
通讯作者：
Mann,KG